Proteasomes are proteolytic machines that are responsible for turnover of the majority of proteins in mammalian cells. The proteasome inhibitor bortezomib (VELCADE) is used for treatment of multiple myeloma, and at least five second-generation proteasome inhibitors including carfilzomib (PR-171) (Demo, et al. (2007) Cancer Res. 67:6383-91; O'Connor, et al. (2009) Clin. Cancer Res. 15:7085-91), NPI-0052 (Chauhan, et al. (2005) Cancer Cell 8:407-19), CEP-18770 (Piva, et al. (2008) Blood 111:2765-75), MLN-9708 (Kupperman, et al. (2010) Cancer Res. 70:1970-80), and ONX-0912 (PR-047)(Zhou, et al. (2009) J. Med. Chem. 52:3028-38) are in clinical testing.
Proteasomes have three different types of active sites, chymotrypsin-like (β5), trypsin-like (β2), and caspase-like (β1). Cells of the immune system express γ-interferon inducible immunoproteasomes, which have slightly different catalytic subunits, namely the β5i (LMP7), β2i (MECL1), and β1i (LMP2). Of these, the chymotrypsin-like sites (β5 and β5i) have long been considered as the only suitable targets for drug development. Bortezomib and all drugs presently undergoing trials were developed to target these sites (Adams (2004) Cancer Cell 5:417-21). However, bortezomib, CEP-18770, and MLN-9708 co-target the caspase-like sites (Piva, et al. (2008) supra; Kupperman, et al. (2010) supra; Kisselev, et al. (2006) J. Biol. Chem. 281:8583-8590; Altun, et al. (2005) Cancer Res. 65:7896-901; Berkers, et al. (2005) Nat. Methods 2:357-62), whereas NPI-0052 co-targets trypsin-like and caspase-like sites (Chauhan, et al. (2005) supra). This raises the question of whether inhibition of these sites is important for the anti-neoplastic activity of these drugs. It has been demonstrated that, in most multiple myeloma cell lines, cytotoxicity of inhibitors does not correlate with inhibition of the chymotrypsin-like sites but does correlate with loss of specificity and onset of inhibition of the trypsin-like sites (Britton, et al. (2009) Chem. Biol. 16:1278-89). These data strongly suggest that the trypsin-like sites are important co-targets for anti-neoplastic agents (Britton, et al. (2009) supra). Cell-permeable inhibitors of these sites are needed to test this hypothesis.
Conventional efforts to develop specific inhibitors of the trypsin-like site have met with limited success to date. Most proteasome inhibitors are short N-terminally capped peptides with an electrophilic group at the C-terminus. This electrophile interacts, reversibly or irreversibly, with the catalytic N-terminal threonine of the proteasome active site. The peptide moiety of the inhibitor binds to the substrate binding pocket of the active site and is largely responsible for the specificity (Kisselev & Goldberg (2001) Chem. Biol. 8:739-758; Groll & Huber (2004) Biochim. Biophys. Acta 1695:33-44), although the specificity may be influenced by an electrophile (Screen, et al. (2010) J. Biol. Chem. 285:40125-40134). The trypsin-like sites cleave peptide bonds after a basic residue and it has been shown that the P3 substituent (Arg) of the β2 selective inhibitor Ac-Tyr-Arg-Leu-Asn-VS (1, SEQ ID NO:1) is of importance in selectivity enhancement (Harris, et al. (2001) Chem. Biol. 8:1131-41; Nazif & Bogyo (2001) Proc. Natl. Acad. Sci. USA 98:2967-2972; Groll, et al. (2002) Chem. Biol. 9:655-62). However, inclusion of basic residues in the P1 and P3 positions are challenging to synthesize and would be expected to render the inhibitor cell-impermeable. In this respect, the few β2-specific aldehydes (Loidl, et al. (1999) Chem. Biol. 6:197-204) and vinyl sulfones (Nazif & Bogyo (2001) supra; Groll, et al. (2002) supra) are not cell-permeable. A cell-permeable peptide vinyl ester (ve), HMB-VSL-VE 2, has been suggested to be a specific inhibitor of the trypsin-like sites (Marastoni, et al. (2005) J. Med. Chem. 48:5038-42; Baldisserotto, et al. (2007) Eur. J. Med. Chem. 42:586-592), but does not show inhibitory activity in conventional assays (Screen, et al. (2010) supra).
